Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Simulation of Spontaneous Ca Oscillations in Astrocytes Mediated by Voltage-Gated Calcium Channels

The purpose of this computational study was to investigate the possible role of voltage-gated Ca²⁺ channels in spontaneous Ca²⁺ oscillations of astrocytes. By incorporating different types of voltage-gated Ca²⁺ channels and a previous model, this study reproduced typical Ca²⁺ oscillations in silico. Our model could mimic the oscillatory phenomenon under a wide range of experimental conditions, including resting membrane potential (−75 to −60 mV), extracellular Ca²⁺ concentration (0.1 to 1500 μM), temperature (20 to 37°C), and blocking specific Ca²⁺ channels. By varying the experimental conditions, the amplitude and duration of Ca²⁺ oscillations changed slightly (both <25%), while the frequency changed significantly (∼400%). This indicates that spontaneous Ca²⁺ oscillations in astrocytes might be an all-or-none process, which might be frequency-encoded in signaling. Moreover, the properties of Ca²⁺ oscillations were found to be related to the dynamics of Ca²⁺ influx, and not only to a constant influx. Therefore, calcium channels dynamics should be used in studying Ca²⁺ oscillations. This work provides a platform to explore the still unclear mechanism of spontaneous Ca²⁺ oscillations in astrocytes.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca²⁺] in the vicinity of plasmalemmal Ca²⁺ channels due to Ca²⁺ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca²⁺] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca²⁺ syntillas. Ca²⁺ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca²⁺ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca²⁺ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca²⁺: one due to influx from the cell exterior through voltage-gated Ca²⁺ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca²⁺ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca²⁺ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.     

Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca²⁺ stores that release Ca²⁺ in response to the second messengers IP₃ and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca²⁺ released from acidic organelles by NAADP subsequently recruits IP₃ or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca²⁺ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca²⁺ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca²⁺-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca²⁺ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca²⁺ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca²⁺ oscillations and waves.     

CamelliA-based simultaneous imaging of Ca2+ dynamics in subcellular compartments

As a universal second messenger, calcium (Ca²⁺) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca²⁺ signature is hampered by limited tools for simultaneously monitoring dynamic Ca²⁺ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca²⁺ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators. We uncovered several Ca²⁺ signatures in three types of Arabidopsis cells in response to internal and external cues, including rapid oscillations of cytosolic Ca²⁺ and apical plasma membrane Ca²⁺ influx in fast-growing Arabidopsis pollen tubes, the spatiotemporal relationship of Ca²⁺ dynamics in four subcellular compartments of root epidermal cells challenged with salt, and a shockwave-like Ca²⁺ wave propagating in laser-wounded leaf epidermis. These observations serve as a testimony to the wide applicability of the CamelliA lines for elucidating the subcellular sources contributing to the Ca²⁺ signatures in plants.

A toolset for simultaneous imaging of Ca²⁺ dynamics in subcellular compartments has uncovered unrecognized Ca²⁺ signatures in Arabidopsis cells in response to developmental and external cues.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

A model of intracellular Ca2+ oscillations based on the activity of the intermediate-conductance Ca2+-activated K+ channels

Intracellular Ca2+ oscillations are observed in a large number of non-excitable cells. While most appear to reflect an intermittent Ca2+ release from intracellular stores, in some instances intracellular Ca2+ oscillations strongly depend on Ca2+ influx, and are coupled to oscillations of the membrane potential, suggesting that a plasma membrane-based mechanism may be involved. We have developed a theoretical model for the latter type of intracellular Ca2+ oscillations based on the Ca2+-dependent modulation of the intermediate-conductance, Ca2+-activated K+ (IKCa) channel. The functioning of this model relies on the Ca2+-dependent activation, and the much slower Ca2+-dependent rundown of this channel. We have shown that Ca2+-dependent activation of the IKCa channels, the consequent membrane hyperpolarization and the resulting increase in Ca2+ influx may confer the positive feedback mechanism required for the ascending phase of the oscillation. The much slower Ca2+-dependent rundown process will conversely halt this positive loop, and establish the descending phase of the intracellular Ca2+ oscillation. We found that this simple model gives rise to intracellular Ca2+ oscillations when using physiologically reasonable parameters, suggesting that IKCa channels could participate in the generation of intracellular Ca2+ oscillations.     

Ca2+ signals and T lymphocytes; "New mechanisms and functions in Ca2+ signalling"

Abstract Interaction between a T cell and an antigen‐presenting cell leads to the rapid formation of an immunological synapse allowing antigen detection by the T cell and the development of an immune response. Antigen detection triggers various cellular responses including a modest but sustained T cell Ca²⁺ increase. In this review are discussed a series of related questions. What are the various molecular events by which a T cell Ca²⁺ response can be triggered in the immunological synapse by a very small amount of antigen ? How is Ca²⁺ released from intracellular stores and how can these stores remain empty for hours ? Through which channels does Ca²⁺ influx takes place, and how is Ca²⁺ influx coupled to Ca²⁺ release from intracellular stores ? What are the main immediate and indirect cellular targets of the Ca²⁺ increase ?     

Synaptic NMDA Receptor-Dependent Ca2+ Entry Drives Membrane Potential and Ca2+ Oscillations in Spinal Ventral Horn Neurons

During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca²⁺-mediated intrinsic membrane potential oscillations in central pattern generator (CPG) neurons. NMDA receptor-dependent intrinsic oscillations require Ca²⁺-dependent K⁺ (K_Ca2) channels for burst termination. However, the location of Ca²⁺ entry mediating K_Ca2 channel activation, and type of Ca²⁺ channel – which includes NMDA receptors and voltage-gated Ca²⁺ channels (VGCCs) – remains elusive. NMDA receptor-dependent Ca²⁺ entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca²⁺ entry is not similarly constrained. Where Ca²⁺ enters relative to K_Ca2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca²⁺ permeating NMDA receptors is the dominant source of Ca²⁺ during NMDA-dependent oscillations in lamprey spinal neurons. This Ca²⁺ entry is synaptically located, NMDA receptor-dependent, and sufficient to activate K_Ca2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca²⁺ entry but leaves membrane potential and Ca²⁺ oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca²⁺ chelation. Taken together, these results demonstrate that K_Ca2 channels are closely located to NMDA receptor-dependent Ca²⁺ entry. The close spatial relationship between NMDA receptors and K_Ca2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses.     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries

The role of vascular gap junctions in the conduction of intercellular Ca²⁺ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca²⁺ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca²⁺ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K⁺ channels with TEA and activating L-type voltage-gated Ca²⁺ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca²⁺ waves and vasoconstriction that spread along an entire artery segment 3000 μm long. Ca²⁺ waves spread as nifedipine-sensitive Ca²⁺ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca²⁺ and vasoconstriction spread at circa 3 mm s⁻¹ and were independent of the endothelium. The spread but not the generation of Ca²⁺ spikes was reversibly blocked by the gap junction inhibitor 18β-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca²⁺-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.     

Ca2+-dependent and Ca2+-independent somatic release from trigeminal neurons

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca²⁺ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca²⁺ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca²⁺ influx, and a slower component triggered by Ca²⁺ influx. The Ca²⁺-dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca²⁺ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca²⁺ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca²⁺ increase generated by an interplay between ER-Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca²⁺ depletion and binds Orai1 to activate Ca²⁺ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca²⁺ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca²⁺ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca²⁺ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca²⁺ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca²⁺ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca²⁺ influx.     

Protein kinase C activator PMA reduces the Ca2+ response to antigen stimulation of adherent RBL-2H3 mucosal mast cells by inhibiting depletion of intracellular Ca2+ stores

Activation of protein kinase C has been shown to reduce the Ca²⁺ responses of a variety of cell types. In most cases, the reduction is due to inhibition of Ca²⁺ influx, but acceleration of Ca²⁺ efflux and inhibition of Ca²⁺ store depletion by protein kinase C activation have also been described. For adherent RBL-2H3 mucosal mast cells, results from whole-cell patch clamp experiments suggest that protein kinase C activation reduces Ca²⁺ influx, while experiments with intact, fura-2-loaded cells suggest that Ca²⁺ influx is not affected. Here we present single-cell data from Ca²⁺ imaging experiments with adherent RBL-2H3 cells, showing that antigen-stimulated Ca²⁺ responses of phorbol 12-myristate 13-acetate (PMA)-treated cells are more transient than those of control cells. PMA also reduced the response to antigen in the absence of extracellular Ca²⁺, indicating that depletion of intracellular Ca²⁺ stores is inhibited. If PMA was added after stores had been depleted by thapsigargin, a small decrease in [Ca²⁺]_i was observed, consistent with a slight inhibition of Ca²⁺ influx. However, the major effect of PMA on the antigen-stimulated Ca²⁺ response is to inhibit depletion of intracellular Ca²⁺ stores. We also show that inhibition of protein kinase C did not enhance the Ca²⁺ response to antigen, suggesting that inhibition of the Ca²⁺ response by activation of protein kinase C does not contribute to the physiological response to antigen. J. Cell. Physiol. 181:113–123, 1999. © 1999 Wiley-Liss, Inc.